Summary: Polysialic acids are synthesized by pathogenic bacteria as capsular polysaccharides. These polymers have been implicated in the virulence of some strains of Escherichia coli, which cause neonatal meningitis and urinary tract infections. Polysialic acid is also present in the developing human brain. There has been significant progress in identifying the gene necessary for capsular polysaccharide biosynthesis in gram negative bacteria. The objective of this project is to determine the mechanism of capsular polysaccharide biosynthesis in virulent encapulated bacteria. Our approach is to characterize the structure and function of the enzymes in the pathway and to use them as tools for understanding sialylation in bacteria and humans. Much of the enzymology of polysialic acid capsular polysaccharide synthesis has been done with the a(2-8) polysialyltransferase complex of E. coli K1. Bacteria containing DNA fragments encoding several capsule related genes have been used as a source of enzyme activity. As a model system for investigating the mechanism of capsular glycosyltransferases we have chosen to investigate the K92 a(2-8)(2-9) polysialyltransferase in a genetic background lacking other capsule related genes. The neuS gene encodes this glycosyltransferase and is the only glycosyltransferase to date identified with synthesis of this polymer. We have shown that the K92 neuS gene product can synthesize both a(2,8) and a(2,9) neuNAc linkages in vitro in a background free of other capsule related gene products and confirmed in vivo synthesis of this polymer by 13C-NMR. The K92 neuS polysialyltransferase is associated with the membrane in lysates of cells harboring the neuS gene in expression vectors, but is not active in a detergent solubilized form suggesting that K92 polysialyltransferase requires association with a membrane for activity. Although the enzyme can transfer sialic acid to the nonreducing end of oligosaccharides with either linkage it is unable to initiate chain synthesis without exogenously added polysialic acid. Thus, the polysialyltransferases encoded by neuS is not sufficient for de novo synthesis of polysaccharide but requires another membrane component for initiation. The acceptor specificity of this polysialyltransferase was studied using sialic acid oligosaccharides of various structures as exogenous acceptors. The enzyme can transfer to the non reducing end of all bacteria polysialic acids, but has a definite preference of a(2,8) acceptors. The minimum acceptor is an oligosaccharide containing a disaccharide of neuNAc a(2,8)neuNAc. Gangliosides containing this disialylated oligosaccharide are elongated, while monsialylated gangliosides are not. Disialylgangliosides are better acceptors than short oligosaccharides suggesting a role for lipid in the elongation reaction. The idea of a lipid linked acceptor is further supported by the elongation of a disialyloligosaccharide possessing a hydrophobic aglycon. Since the neuS gene product is not sufficient for initiation of polysaccharide synthesis we used complementation experiments to determine the minimum number of proteins encoded by the K92 gene cluster that are required for synthesis of polysialic acid in the absence of exogenously added acceptor. The capsule specific genes in region 2, neuDBACES are not sufficient to support initiation, suggesting that adjacent kps genes may also be reguired. This would require that perhaps a complex of proteins are involved in the polymerization of polysialic acid. To address this we have used readiation traget analysis in collaboration with Dr. Ellis Kempner in the Laboratory of Physical Biology, NIAMD, NIH. Using this technique were have estimated an approximate size for the catalytic activity forming polysialic acid in the presence and absence of exogenous acceptor. These size responsible fr the activities assayed under these conditions do not differ significantly suggesting that a large complex is probobably not required. The information learned in the investigation of the pathway for synthesis of polysialic acid the absence of exogenously added acceptor. capsules has been used in a collaboration with a group whose objective is to construct an insect expression system capable of producing sialylated glycoproteins. Sialic acid synthetase from human, Campylobacter jejuni, and drosophilla have been expressed in E. coli and purified. A CMP-siaic acid synthetase has been cloned from a human gene bank and characterized based on its similarity to the bacteria enzyme.